Polypeptide expressed in the horny layer of epidermis and use thereof

ABSTRACT

The present invention relates to a cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, a purified natural or synthetic epidermis-specific polypeptide which is involved in horny layer cell cohesion. The invention also relates to a cosmetic or pharmaceutical composition comprising a mixture of polypeptides derived from the proteolysis of the purified polypeptide of the present invention. The present invention further relates to a method for strengthening horny layer cell cohesion which comprises applying the cosmetic or pharmaceutical composition of the present invention to the skin. Finally, the present invention relates to a method for reducing horny layer cell cohesion, and thereby promoting exfoliation, wherein said method comprises applying to the skin a cosmetic or pharmaceutical composition comprising an effective amount of at least one protease active on the polypeptide of the present invention.

The subject of the invention is a cosmetic or pharmaceutical compositioncomprising, in a physiologically acceptable medium, at least onepurified natural or synthetic polypeptide, specific to the epidermis,having a role in intercorneocyte cohesion. The subject of the inventionis also a cosmetic or pharmaceutical composition comprising a mixture ofpolypeptides derived from the proteolysis of the purified polypeptide, amethod of cosmetic treatment for strengthening intercorneocyte cohesionand a method of cosmetic treatment for reducing intercorneocytecohesion, and therefore for promoting desquamation.

Human skin consists of two compartments, namely a deep compartment, thedermis, and a superficial compartment, the epidermis.

The dermis provides the epidermis with a solid support. It is also itsfeeder component. It consists mainly of fibroblasts and an extracellularmatrix itself mainly composed of collagen, elastin and a substancecalled ground substance, these components being synthesized by thefibroblast. Leukocytes, mastocytes or tissue macrophages are also foundtherein. It also consists of blood vessels and of nerve fibers.

Natural human epidermis is composed mainly of three types of cells whichare the keratinocytes, which are highly predominant, the melanocytes andthe Langerhans' cells. Each of these cell types contributes, through itsspecific functions, to the essential role played by the skin in theorganism.

The epidermis is conventionally divided into a basal layer ofkeratinocytes which constitutes the germinative layer of the epidermis,a so-called prickle cell layer consisting of several layers ofpolyhedral cells arranged on the germinative cells, a so-called granularlayer consisting of flattened cells containing distinct cytoplasmicinclusions, the keratohyalin granules, and finally a top layer calledhorny layer (or stratum corneum), consisting of keratinocytes at thefinal stage of their differentiation, called corneocytes. These areanucleated, mummified cells which are derived from the keratinocytes.

The corneocytes are mainly composed of a fibrous matrix containingcytokeratins, surrounded by a very resistant structure 15 nm thick,called horny or hornified envelope. The stacking of these corneocytesconstitutes the horny layer which is responsible for the barrierfunction of the epidermis. During the normal desquamation process, themost superficial corneocytes become detached from the surface of theepidermis.

Intercellular structures derived from the desmosomes, called corneosomesor corneodesmosomes, have been described in the horny layer. Recentstudies have shown their key importance in intercorneocyte cohesion aswell as in the desquamation process. In particular, a close correlationexists between cell dissociation and proteolysis of certaincorneodesmosomal components such as desmoglein I.

Several serine proteases of the trypsin or chymotrypsin type appear tobe involved in the proteolysis of the corneodesmosomes, in particularthe chymotryptic enzyme of the horny layer (stratum corneum chymotrypticenzyme).

Numerous pathological conditions of the skin are characterized by theproduction of a thick horny layer and by an abnormal desquamation, thatis to say by hyperkeratosis. The latter may occur on any anatomical skinarea and in a wide variety of clinical contexts. Its physiopathologicalsubstratum and its cause are varied.

By way of example, there may be mentioned:

xerosis (or dryness of the skin),

ichthyoses,

psoriasis,

certain benign or malignant tumour lesions,

reactive hyperkeratoses.

Other pathological conditions are characterized by transdifferentiationor metaplasia, at the level of the mucosae, Malpighian or otherwise, butnormally nonhornified, which become hornified, that is to say whichbecome covered with an abnormal epithelium, producing a horny layer atits surface. Although the genital mucosae and those of the upperaerodigestive tracts are most often involved, these metaplasias may beseated in various anatomical areas.

By way of examples, there may be mentioned

leukokeratosis of the uterine neck during prolapsus,

buccal leukokeratoses,

keratotic benign tumour lesions of the Malpighian mucosae

By contrast, some pathological manifestations cause thinning of theepidermis and in particular of the horny layer, resulting in excessivefragility of the skin covering. It may be seated in various anatomicalareas, its cause is variable and it may be constitutional or acquired.

By way of examples, there may be mentioned:

trophic skin disorders of the lower limbs in patients carrying vascularpathological conditions: varicose veins, arteriopathies (diabetes,arteriosclerosis and the like),

trophic skin disorders in the context of an algodystrophic syndrome,

trophic disorders following abnormal cicatrization.

The purification and knowledge of the polypeptides involved inintercorneocyte cohesion is one of the routes which could allow theproduction of products for combating the effects of an excess or adeficiency of polypeptides of this type, in particular at the surface ofthe skin.

One of the objects of the invention is to provide a compositioncomprising a polypeptide involved in intercorneocyte cohesion inpurified form.

After long and laborious studies because of its low representation amongthe proteins of the epidermis, and its high instability and its highsensitivity to proteases, the applicant has identified, isolated andpurified by biochemical techniques, from a human epidermis, apolypeptide specific to the hornified epithelia. This polypeptide, whichwill also be called elsewhere in the text “corneodesmosine” is expressedin the horny layer of the epidermis and is involved in intercorneocytecohesion. The applicant has determined the primary amino acid sequencethereof.

The subject of the invention is therefore a cosmetic or pharmaceuticalcomposition comprising, in a physiologically acceptable medium, at leastone purified natural or synthetic polypeptide, the said polypeptidebeing characterized in that it corresponds to the following amino acidsequence SEQ ID NO: 1:

Met Gly Ser Ser Arg Ala Pro Trp Met Gly Arg Val Gly Gly His Gly 1               5                  10                  15 Met Met AlaLeu Leu Leu Ala Gly Leu Leu Leu Pro Gly Thr Leu Ala            20                  25                  30 Lys Ser Ile GlyThr Phe Ser Asp Pro Cys Lys Asp Pro Thr Arg Ile        35                  40                  45 Thr Ser Pro Asn AspPro Cys Leu Thr Gly Lys Gly Asp Ser Ser Gly    50                  55                  60 Phe Ser Ser Tyr Ser GlySer Ser Ser Ser Gly Ser Ser Ile Ser Ser65                  70                  75                  80 Ala ArgSer Ser Gly Gly Gly Ser Ser Gly Ser Ser Ser Gly Ser Ser                85                  90                  95 Ile Ala GlnGly Gly Ser Ala Gly Ser Phe Lys Pro Gly Thr Gly Tyr            100                 105                 110 Ser Gln Val SerTyr Ser Ser Gly Ser Gly Ser Ser Leu Gln Gly Ala        115                 120                 125 Ser Gly Ser Ser GlnLeu Gly Ser Ser Ser Ser His Ser Gly Ser Ser    130                 135                 140 Gly Ser His Ser Gly SerSer Ser Ser His Ser Ser Ser Ser Ser Ser145                 150                 155                 160 Phe GlnPhe Ser Ser Ser Ser Phe Gln Val Gly Asn Gly Ser Ala Leu                165                 170                 175 Pro Thr AsnAsp Asn Ser Tyr Arg Gly Ile Leu Asn Pro Ser Gln Pro            180                 185                 190 Gly Gln Ser SerSer Ser Ser Gln Thr Ser Gly Val Ser Ser Ser Gly        195                 200                 205 Gln Ser Val Ser SerAsn Gln Arg Pro Cys Ser Ser Asp Ile Pro Asp    210                 215                 220 Ser Pro Cys Ser Gly GlyPro Ile Val Ser His Ser Gly Pro Tyr Ile225                 230                 235                 240 Pro SerSer His Ser Val Ser Gly Gly Gln Arg Pro Val Val Val Val                245                 250                 255 Val Asp GlnHis Gly Ser Gly Ala Pro Gly Val Val Gln Gly Pro Pro            260                 265                 270 Cys Ser Asn GlyGly Leu Pro Gly Lys Pro Cys Pro Pro Ile Thr Ser        275                 280                 285 Val Asp Lys Ser TyrGly Gly Tyr Glu Val Val Gly Gly Ser Ser Asp    290                 295                 300 Ser Tyr Leu Val Pro GlyMet Thr Tyr Ser Lys Gly Lys Ile Tyr Pro305                 310                 315                 320 Val GlyTyr Phe Thr Lys Glu Asn Pro Val Lys Gly Ser Pro Gly Val                325                 330                 335 Pro Ser PheAla Ala Gly Pro Pro Ile Ser Glu Gly Lys Tyr Phe Ser            340                 345                 350 Ser Asn Pro IleIle Pro Ser Gln Ser Ala Ala Ser Ser Ala Ile Ala        355                 360                 365 Phe Gln Pro Val GlyThr Gly Gly Val Gln Leu Cys Gly Gly Gly Ser    370                 375                 380 Thr Gly Ser Lys Gly ProCys Ser Pro Ser Ser Ser Arg Val Pro Ser385                 390                 395                 400 Ser SerSer Ile Ser Ser Ser Ala Gly Ser Pro Tyr His Pro Cys Gly                405                 410                 415 Ser Ala SerGln Ser Pro Cys Ser Pro Pro Gly Thr Gly Ser Phe Ser            420                 425                 430 Ser Ser Ser SerSer Gln Ser Ser Gly Lys Ile Ile Leu Gln Pro Cys        435                 440                 445 Gly Ser Lys Ser SerSer Ser Gly His Pro Cys Met Ser Val Ser Ser    450                 455                 460 Leu Thr Leu Thr Gly GlyPro Asp Gly Ser Pro His Pro Asp Pro Ser465                 470                 475                 480 Ala GlyAla Lys Pro Cys Gly Ser Ser Ser Ala Gly Lys Ile Pro Cys                485                 490                 495 Arg Ser IleArg Asp Ile Leu Ala Gln Val Lys Pro Leu Gly Pro Gln            500                 505                 510 Leu Ala Asp ProGlu Val Phe Leu Pro Gln Gly Glu Leu Leu Asp Ser        515                 520                 525 Pro

The polypeptide of the invention may be of natural or synthetic origin.Synthetic is understood here to mean any polypeptide obtained chemicallyor by production in an organism after introducing into this organism thecomponents necessary for this production.

The polypeptide of the invention may be derived from any possibleorigin, namely either animal, in particular mammalian and still moreparticularly human, origin or plant origin or from microorganisms (interalia viruses, phages or bacteria) or from fungi, without prejudging thefact that it is present naturally or otherwise in the said organism oforigin.

Preferably, the polypeptide of the invention is of natural origin,purified from mammalian tissues, particularly from mammalian skin.

Preferably, the polypeptide of the invention is purified from human skinand still more preferably from human epidermis.

As indicated above, intercorneocyte cohesion is apparently due, interalia, to the existence, in the horny layer, of polypeptides specific tothe structures involved in the intercorneocyte junction.

Accordingly, the polypeptide of the invention is specific to the hornylayer and to the granular layer and, preferably, the polypeptideaccording to the invention is specific to the structures involved in theintercorneocyte junction, particularly of the corneodesmosomes.

It is known, in general, that the mature polypeptides which are found incells are derived from the maturation of precursors which contain, intheir sequence, the sequence of the mature polypeptide.

Accordingly, the invention also relates to a cosmetic or pharmaceuticalcomposition comprising, in a physiologically acceptable medium, anypolypeptide whose sequence partly consists of the sequence of thepolypeptide of the invention.

It is also known that the polypeptides may undergo post-translationalmodifications such as the formation of disulphide bonds, specificproteolytic cleavages, the addition of carbohydrates (glycosylation),phosphorylation, in particular at the level of the serines and/or of thethreonines and/or of the tyrosines, and/or combination with lipids.

The invention therefore relates to a cosmetic or pharmaceuticalcomposition comprising, in a physiologically acceptable medium, at leastone polypeptide of the invention which has undergone post-translationalmodifications or not.

The polypeptide of the invention may have undergone one or morepost-translational modifications.

Preferably, the polypeptide according to the invention is glycosylatedand/or phosphorylated.

It is known to classify polypeptides according to their isoelectricpoint. Preferably, the polypeptide of the invention is basic.

Of course the primary amino acid sequence as well as the variouspost-translational modifications undergone by the polypeptide areresponsible for the fact that the said polypeptide may be characterizedby its molecular weight, expressed in kilodaltons.

The polypeptide of the invention has an apparent molecular weight,determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), ofbetween 50 and 60 kilodaltons, preferably between 52 and 56 kilodaltons.

Most preferably, the polypeptide of the invention is a basic,phosphorylated, glycosylated polypeptide having an apparent molecularweight of between 50 and 60, preferably between 52 and 56 kilodaltons.

Thus preferably, the subject of the invention is a cosmetic orpharmaceutical composition comprising, in a physiologically acceptablemedium, at least one polypeptide of the invention, the said polypeptidebeing phosphorylated, basic and glycosylated and having an apparentmolecular weight of between 50 and 60 kilodaltons, preferably between 52and 56 kilodaltons.

It is also known that the primary amino acid sequence of a polypeptidedetermines sites specifically recognized by proteases which, once therecognition of these sites has been achieved, will, with or withoutattachment to the said polypeptide, induce its cleavage by proteolysis.

Accordingly, the invention also relates to a cosmetic or pharmaceuticalcomposition comprising, in a physiologically acceptable medium, at leastone mixture of polypeptides derived from the proteolysis of thepolypeptide of the invention.

As described above, some pathological conditions may be due to excessivedesquamation which may be assumed to be due to a deficiency ofpolypeptides involved in intercorneocyte cohesion.

Some pathological manifestations cause thinning of the epidermis and, inparticular, of the horny layer, resulting in excessive fragility of theskin covering, which may be seated in any anatomical skin area and mayhave various causes, constitutional or acquired.

Accordingly, the composition according to the invention is intended fortreating the thinning of the epidermis, and in particular the hornylayer, and/or for treating excessive fragility of the skin coveringand/or for strengthening intercorneocyte cohesion and/or inducing thethickening of the horny layer.

Likewise, the composition according to the invention is intended fortreating the thinning of the epidermis, and in particular of the hornylayer and/or for treating excessive fragility of the skin coveringand/or for strengthening intercorneocyte cohesion and/or inducing thethickening of the horny layer.

Likewise, the composition of the invention may be used for causinglocalized thickening of the horny layer at the level of the skin regionswhich have to be subjected to repeated microtraumas.

By way of example, there may be mentioned the preventive treatment ofsubepidermal blisters (“ampullae”) in sportspeople.

The subject of the invention is also a method of cosmetic treatment fortreating the thinning of the epidermis, and in particular of the hornylayer, and/or for treating excessive fragility of the skin coveringand/or for strengthening intercorneocyte cohesion and/or inducing thethickening of the horny layer, characterized in that a cosmeticcomposition according to the invention is applied to the skin of thesubject to be treated.

The subject of the invention is also a method of cosmetic treatment fortreating trophic skin disorders, for example of patients carryingvascular pathological conditions such as varicose veins orarteriopathies (diabetes, arteriosclerosis and the like), trophic skindisorders of patients carrying algodystrophic syndromes or thosefollowing cicatrization disorders.

Analysis of the primary amino acid sequence of the protein according tothe invention shows that it has recognition and binding sites for knownproteases or specific sites for cleavage by chemical agents. There maybe mentioned, as example, the sites for Chymotrypsin, Cathepsin,proteinase K, Subtilisin, protease V8, Thermolysin, Thrombin, Trypsin,Papain, Pepsin, Proline-Endopeptidase, Endoproteinase GluC,Endoproteinase LysC, Endoproteinase AspN, Endoproteinase ArgC(Clostripain), Myxobacter AL117, Elastase, chymotryptic enzyme of thehorny layer, cyanogen bromide, N-chlorosuccinimide or specific sites foracid hydrolysis (70% formic acid, 7 M Guanidine-HCl, 400° C., 24 h).

Intercorneocyte cohesion is apparently due to the existence, in thehorny layer, of polypeptides soecific to the structures involved in theintercorneocyte junction such as, in particular, the polypeptide of theinvention. It has been seen that certain hyperkeratotic pathologicalconditions could be linked to an excessive intercorneocyte cohesion, duein particular to the polypeptide of the invention.

Accordingly, the subject of the invention is a cosmetic orpharmaceutical composition comprising, in a physiologically acceptablemedium, an effective quantity of at least one protease active on thepolypeptide of the invention, chosen from Endoproteinase LysC,Endoproteinase GluC, Proline-Endopeptidase, Thrombin, Pepsin, MyxobacterAL117 and Elastase.

These proteases may be isolated from plants, animals, bacteria, virusesor fungi.

The polypeptide of the invention may be glycolysed. Accordingly, thecomposition as defined above may contain, in addition, a glycosidasewhich may be chosen from those isolated from plants, animals, fungi ormicroorganisms, in particular bacteria. There may be mentioned, by wayof example, neuraminidases, mannosidases, galactosidases, glucosidases,N-acetylglucosaminidases and N-acetylgalactos aminidases.

As seen above, some pathological conditions are characterized by theproduction of a thickened epidermal horny layer and by abnormaldesquamation, that is to say by hyperkeratosis which may occur in anyanatomical skin area, in various clinical contexts and whosephysiopathological substratum and cause may be varied.

The invention also relates to a cosmetic or pharmaceutical compositioncomprising a protease, as defined above, for treating, for example,hyperkeratosis, xerosis (or dryness of the skin), ichthyoses, psoriasis,certain benign or malignant hyperkeratotic tumour lesions, and reactivekeratoses.

The invention also relates to a cosmetic or pharmaceutical compositioncomprising a protease, as defined above, for treating pathologicalconditions which are characterized by transdifferentiation ormetaplasia, at the level of mucosae, Malpighian or otherwise, butnormally nonhornified, which become hornified such as, for example,leukokeratosis of the uterine neck during prolapsus, buccalleukokeratoses, or benign or malignant hyperkeratotic tumour lesions ofthe Malpighian mucosae.

Another object of the invention is to provide a method of cosmetictreatment for combating excessive intercorneocyte cohesion and thereforefor increasing desquamation and in particular the excesses due to thepolypeptide of the invention, which method consists in applying to theskin a cosmetic composition comprising at least one protease having aspecific recognition and/or binding and cleavage site within the primaryamino acid sequence of the polypeptide of the invention.

It is known that a protein is synthesized in the cells from a templateof deoxyribonucleic acids encoding the said protein. It is also knownthat the genetic code is degenerate. Accordingly, the amino acidsequence of the polypeptide of the invention may be derived from variousnatural or synthetic deoxyribonucleic acid sequences. Syntheticdeoxyribonucleic acid sequence is understood here to mean any sequenceobtained chemically or by genetic engineering.

The said deoxyribonucleic acid sequences may be derived from anypossible origin, namely either animal, in particular mammalian and stillmore particularly human origin or plant origin or from microorganisms(inter alia viruses, phages, or bacteria) or from fungi, withoutprejudging the fact that they are present naturally or otherwise in thesaid organism of origin.

The applicant has isolated, purified and sequenced, using molecularbiology techniques, in particular the screening of libraries forexpression of complementary deoxyribonucleic acids prepared from humanepidermis, a deoxyribonucleic acid fragment encoding the polypeptide ofthe invention.

The subject of the invention is also therefore a cosmetic orpharmaceutical composition comprising, in a physiologically acceptablemedium, any deoxyribonucleic acid sequences, natural or synthetic, allor part of which encodes the primary amino acid sequence of thepolypeptide of the invention.

During these studies, the applicant has been able to isolate and purifya deoxyribonucleic acid sequence encoding the primary amino acidsequence of the polypeptide of the invention from human skin.

Particularly, the subject of the invention is a cosmetic orpharmaceutical composition comprising, in a physiologically acceptablemedium, an isolated deoxyribonucleic acid fragment comprising at leastthe following coding nucleotide sequence SEQ ID NO: 2:

1 ATG GGC TCG TCT CGG GCA CCC TGG ATG GGG CGT GTG GGT GGG CAC 46 GGG ATGATG GCA CTG CTG CTG GCT GGT CTC CTC CTG CCA GGG ACC 91 TTG GCT AAG AGCATT GGC ACC TTC TCA GAC CCC TGT AAG GAC CCC 136 ACG CGT ATC ACC TCC CCTAAC GAC CCC TGC CTC ACT GGG AAG GGT 181 GAC TCC AGC GGC TTC AGT AGC TACAGT GGC TCC AGC AGT TCT GGC 226 AGC TCC ATT TCC AGT GCC AGA AGC TCT GGTGGT GGC TCC AGT GGT 271 AGC TCC AGC GGA TCC AGC ATT GCC CAG GGT GGT TCTGCA GGA TCT 316 TTT AAG CCA GGA ACG GGG TAT TCC CAG GTC AGC TAC TCC TCCGGA 361 TCT GGC TCT AGT CTA CAA GGT GCA TCC GGT TCC TCC CAG CTG GGG 406AGC AGC AGC TCT CAC TCG GGA AGC AGC GGC TCT CAC TCG GGA AGC 451 AGC AGCTCT CAT TCG AGC AGC AGC AGC AGC TTT CAG TTC AGC AGC 496 AGC AGC TTC CAAGTA GGG AAT GGC TCT GCT CTG CCA ACC AAT GAC 541 AAC TCT TAC CGC GGA ATACTA AAC CCT TCC CAG CCT GGA CAA AGC 586 TCT TCC TCT TCC CAA ACC TCT GGGGTA TCC AGC AGT GGC CAA AGC 631 GTC AGC TCC AAC CAG CGT CCC TGT AGT TCGGAC ATC CCC GAC TCT 676 CCC TGC AGT GGA GGG CCC ATC GTC TCG CAC TCT GGCCCC TAC ATC 721 CCC AGC TCC CAC TCT GTG TCA GGG GGT CAG AGG CCT GTG GTGGTG 766 GTG GTG GAC CAG CAC GGT TCT GGT GCC CCT GGA GTG GTT CAA GGT 811CCC CCC TGT AGC AAT GGT GGC CTT CCA GGC AAG CCC TGT CCC CCA 856 ATC ACCTCT GTA GAC AAA TCC TAT GGT GGC TAC GAG GTG GTG GGT 901 GGC TCC TCT GACAGT TAT CTG GTT CCA GGC ATG ACC TAC AGT AAG 946 GGT AAA ATC TAT CCT GTGGGC TAC TTC ACC AAA GAG AAC CCT GTG 991 AAA GGC TCT CCA GGG GTC CCT TCCTTT GCA GCT GGG CCC CCC ATC 1036 TCT GAG GGC AAA TAC TTC TCC AGC AAC CCCATC ATC CCC AGC CAG 1081 TCG GCA GCT TCC TCG GCC ATT GCG TTC CAG CCA GTGGGG ACT GGT 1126 GGG GTC CAG CTC TGT GGA GGC GGC TCC ACG GGC TCC AAG GGACCC 1171 TGC TCT CCC TCC AGT TCT CGA GTC CCC AGC AGT TCT AGC ATT TCC1216 AGC AGC GCC GGT TCA CCC TAC CAT CCC TGC GGC AGT GCT TCC CAG 1261AGC CCC TGC TCC CCA CCA GGC ACC GGC TCC TTC AGC AGC AGC TCC 1306 AGT TCCCAA TCG AGT GGC AAA ATC ATC CTT CAG CCT TGT GGC AGC 1351 AAG TCC AGC TCTTCT GGT CAC CCT TGC ATG TCT GTC TCC TCC TTG 1396 ACA CTG ACT GGG GGC CCCGAT GGC TCT CCC CAT CCT GAT CCC TCC 1441 GCT GGT GCC AAG CCC TGT GGC TCCAGC AGT GCT GGA AAG ATC CCC 1486 TGC CGC TCC ATC CGG GAT ATC CTA GCC CAAGTG AAG CCT CTG GGG 1531 CCC CAG CTA GCT GAC CCT GAA GTT TTC CTA CCC CAAGGA GAG TTA 1576 CTC GAC AGT CCA TAA

The subject of the invention is also a cosmetic or pharmaceuticalcomposition comprising, in a physiologically acceptable medium, a senseor antisense ribonucleic acid sequence corresponding to the saidsequence SEQ ID NO: 2.

Its subject is also the use of the said deoxyribonucleic acid sequencesfor the production of the polypeptide of the invention or of acorresponding ribonucleic acid by any known technique such as forexample synthesis in vitro, from reconstituted media, or synthesis byorganisms.

The subject of the invention is also the use of the polypeptide of theinvention or of its proteolysis fragments, and of any synthetic peptidededuced from its sequence, for preparing or purifying, optionally fromthe epidermis, any molecule, structural or functional, capable ofbinding specifically to the said purified polypeptide or to the saidpurified proteolysis fragments or to the said synthetic peptide. Thismolecule may in particular correspond to other structural proteinsspecific to the corneodesmosomes and various enzymes of the horny layer,of the “protease”, “glycosidase” or “phosphatase” type.

The subject of the invention is also the use of the polypeptide of theinvention or of its proteolytic fragments and of any synthetic peptidededuced from its sequence, for preparing specific monoclonal antibodiesand antisera, intended in particular for purifying this protein and itsfragments. By extension, the subject of the invention is also any use ofthe said sequence for producing recombinant antibodies or antibodyfragments, regardless of the biological system used for producing thelatter.

Whatever their nature, the compositions of the invention may beingested, injected or applied to the skin (over any skin area of thebody) or the mucosae (buccal, jugal, gingival, genital, conjunctival andthe like).

Preferably, the compositions of the invention are applied to the skin orthe mucosae.

Depending on the mode of administration, the compositions according tothe invention may be provided in any of the galenical forms normallyused.

For a topical application to the skin, the composition may take theform, in particular, of an aqueous or oily solution or of a dispersionof the lotion or serum type, of emulsions of liquid or semi-liquidconsistency of the milk type, obtained by dispersion of a fatty phase inan aqueous phase (O/W) or conversely (W/O), or of suspensions oremulsions of soft consistency of the aqueous or anhydrous cream or geltype, or of microcapsules or microparticles, or of vesicular dispersionsof the ionic and/or nonionic type or of foams or alternatively in theform of aerosol compositions also comprising a pressurized propellant.These compositions are prepared according to the customary methods.

For injection, the composition may be provided in the form of an aqueousor oily lotion or in the form of a serum. For the eyes, it may beprovided in the form of drops, and for ingestion, it may be provided inthe form of capsules, granules, syrups or tablets.

The quantities of the various constituents of the compositions accordingto the invention are those conventionally used in the fields considered.

These compositions constitute in particular cleansing, protective,treatment or care creams for the face, for the hands, for the feet, forthe large anatomical folds or for the body, (for example day creams,night creams, make-up removing creams, foundation creams, antisuncreams), fluid foundations, make-up removing milks, protective or carebody milks, antisun milks, skincare lotions, gels or foams, such ascleansing lotions, artisun lotions, artificial tanning lotions, bathcompositions, deodorant compositions containing a bactericidal agent,aftershave gels or lotions, depilatory creams, compositions againstinsect bites, antipain compositions or compositions for treating certainskin diseases such as eczema, rosacea, psoriasis, lichens and severepruritus.

The compositions according to the invention may also consist of solidpreparations constituting cleansing soaps or cakes.

The compositions may also be packaged in the form of an aerosolcomposition also containing a pressurized propellant.

The composition according to the invention may also be a composition forthe care of the scalp, especially a shampoo, a hair setting lotion, atreatment lotion, a hair styling cream or gel, a dyeing (especiallyoxidation dyeing) composition optionally in the form of dyeing shampoos,restructuring lotions for the hair, a permanent-waving composition(especially a composition for the first stage of a permanent waving), alotion or gel against hair loss, an antiparasitic shampoo and the like.

The composition may also be for dentibuccal use, for example atoothpaste. In this case, the composition may contain customaryadjuvants and additives for compositions for buccal use and especiallysurfactants, thickening agents, humectants, polishing agents such assilica, various active ingredients such as fluorides, in particularsodium fluoride, and optionally sweetening agents such as sodiumsaccharinate.

When the composition is an emulsion, the proportion of fatty phase mayrange from 5% to 80% by weight, and preferably from 5% to 50% by weightrelative to the total weight of the composition. The oils, waxes,emulsifiers and coemulsifiers used in the composition in the form of anemulsion are chosen from those conventionally used in the cosmeticfield. The emulsifier and the coemulsifier are present in thecomposition in a proportion ranging from 0.3% to 30% by weight, andpreferably from 0.5 to 20% by weight relative to the total weight of thecomposition. The emulsion may, in addition, contain lipid vesicles.

When the composition is an oily gel or a solution, the fatty phase mayrepresent more than 90% of the total weight of the composition.

In a known manner, the cosmetic composition may also contain adjuvantscommon in the cosmetic field, such as hydrophilic or lipophilic gellingagents, hydrophilic or lipophilic additives, preservatives,antioxidants, solvents, perfumes, fillers, screening agents, odourabsorbers and colouring materials. The quantities of these variousadjuvants are those conventionally used in the cosmetic field, and forexample from 0.01% to 10% of the total weight of the composition. Theseadjuvants, depending on their nature, may be introduced into the fattyphase, into the aqueous phase and/or into the lipid spherules.

As oils or waxes which can be used in the invention, there may bementioned mineral oils (petroleum jelly), vegetable oils (liquidfraction of shea butter, sunflower oil), animal oils (perhydrosqualene),synthetic oils (Purcellin oil), silicone oils or waxes (cyclomethicone)and fluorinated oils (perfluoropolyethers), beeswaxes, carnauba orparaffin waxes. Fatty alcohols and fatty acids (stearic acid) may beadded to these oils.

As emulsifiers which can be used in the invention, there may bementioned for example glycerol stearate, polysorbate 60 and thePEG-6/PEG-32/Glycol Stearate mixture sold under the name Tefose^(R) 63by the company Gattefosse.

As solvents which can be used in the invention, there may be mentionedthe lower alcohols, especially ethanol and isopropanol, and propyleneglycol.

As hydrophilic gelling agents which can be used in the invention, theremay be mentioned the carboxyvinyl polymers (carbomer), acryliccopolymers such as acrylate/alkylacrylate copolymers, polyacrylamides,polysaccharides such as hydroxypropylcellulose, natural gums and clays,and, as lipophilic gelling agents; there may be mentioned modified clayssuch as bentones, metal salts of fatty acids such as aluminium stearatesand hydrophobic silica, ethylcellulose and polyethylene.

The composition may contain other hydrophilic active agents, such asproteins or protein hydrolysates, amino acids, polyols, urea, allantoin,sugars and sugar derivatives, water-soluble vitamins, plant extracts andhydroxy acids.

As lipophilic active agents, there may be used retinol (vitamin A) andits derivatives, tocopherol (vitamin E) and its derivatives, essentialfatty acids, ceramides, essential oils, salicylic acid and itsderivatives.

According to the invention, the composition may combine at least oneextract of at least one Iridaceae with other active agents intendedespecially for the prevention and/or treatment of skin conditions. Amongthese active agents, there may be mentioned, by way of example:

agents reducing skin differentiation and/or proliferation and/orpigmentation such as retinoic acid and its isomers, retinol and itsesters, vitamin D and its derivatives, oestrogens such as oestradiol,kojic acid or hydroquinone;

antibacterials such as clindamycin phosphate, erythromycin orantibiotics of the tetracycline class;

antiparasitic agents, in particular metronidazole, crotamiton orpyrethroids;

antifungal agents, in particular the compounds belonging to theimidazole class such as econazole, ketoconazole or miconazole or theirsalts, the polyene compounds, such as amphotericin B, the compounds ofthe allylamine family, such as terbinafine, or octopirox;

antiviral agents such as acyclovir;

steroidal anti-inflammatory agents such as hydrocortisone, betamethasonevalerate or clobetasol propionate, or nonsteroidal anti-inflammatoryagents such as, for example, ibuprofen and its salts, diclofenac and itssalts, acetylsalicylic acid, acetaminophen or glycyrrhizic acid;

anaesthetic agents such as lidocaine hydrochloride and its derivatives;

antipruritic agents such as thenaldine, trimeprazine or cyproheptadine;

keratolytic agents such as α- and β-hydroxycarboxylic orβ-ketocarboxylic acids, their salts, amides or esters and moreparticularly hydroxy acids such as glycolic acid, lactic acid, salicylicacid, citric acid and, in general, fruit acids and 5-n-octanoylsalicylicacid;

anti-free-radical agents, such as α-tocopherol or its esters, superoxidedismutases, certain metal chelators or ascorbic acid and its esters;

antiseborrhoeic agents such as progesterone;

antidandruff agents such as octopirox or zinc pyrithione;

anti-acne agents such as retinoic acid or benzoyl peroxide.

Accordingly, according to a specific embodiment, the compositionaccording to the invention also comprises at least one agent chosen fromantibacterial, antiparasitic, antifungal, antiviral, anti-inflammatory,antipruritic, anaesthetic, keratolytic, anti-free-radical,antiseborrhoeic, antidandruff and anti-acne agents, and/or agentsreducing skin differentiation and/or proliferation and/or pigmentation.

EXAMPLES Monoclonal Antibodies

The murine monoclonal antibodies G36-19, F28-27 and B17-21 (IgG1) whichare specific for the polypeptide of the invention, corneodesmosine, formpart of a series of antibodies directed against epidermaldifferentiation antigens, produced after immunization of a mouse with ahuman plantar horny layer homogenate, and then characterized. Theascites of the monoclonal antibody MOPC-21 (IgG1) (Sigma Chemical Co.,St Louis, Mo.) was used as negative control. The anti-phosphoserineantibody kit from Biomol Feinchenukalien GmbH (monoclonal antibody 1C8,4A3, 4A9 and 4H4) and the monoclonal antibody PSR45 from Sigma were alsoused.

Isolation and Characterization of the Polypeptide

Sequential Protein Extraction

Starting with mammary skin (obtained from reductive mammaplasty), theepidermis was mechanically separated from the dermis after heattreatment of the skin for 5 minutes at 56° C. in a phosphate buffer, andthen sequentially homogenized, at 4° C., in equal volumes of thefollowing buffers (three times in each buffer: i) TE buffer: 40 mMTris-HCl , pH 7.5; 10 mM ethylenediaminetetraacetate (EDTA); 0.25 mMphenylmethylsulphonyl fluoride and 2 μg/ml of each of the followinginhibitors: aprotinin, pepstatin A and leupeptin; ii) TENP40 buffer: TEcontaining a detergent, Nonidet P-40 at 0.5%; iii) TEU buffers: TEcontaining various concentrations of urea (from 2 to 8 M). After eachextraction, the homogenates were centrifuged for 15 minutes at 15,000 gand the supernatants collected. The first supernatant from eachextraction was kept at −30° C. until the time of use. Finally, thepellet corresponding to the last extraction in the buffer containing 8 Murea was homogenized in TUDTT buffer: 35 mM Tris-HCl, pH 6.8; 8 M urea;50 mM dithiothreitol; 5% glycerol; 0.25 mM phenylmethylsulphonylfluoride and 2 μg/ml of each of the same inhibitors, incubated for 30minutes at 95° C. and centrifuged as above. The protein concentrationswere measured using the Pierce system (Coomassie Plus protein assay,Pierce Chemical Co., Rockford, Ill.).

Electrophoresis of the Proteins and Immunodetection

The proteins were separated by polyacrylamide gel electrophoresis in thepresence of SDS (SDS-PAGE) in 7.5 or 10% acrylamide gels or bytwo-dimensional electrophoresis in the presence of urea, using thePharmacia system (PhastSystem™; Pharmacia LKB). An isoelectric focusing(IEF) or a non-equilibrium pH gel electrophoresis (NEpHGE) was carriedout in the first dimension, and then an SDS-PAGE was carried out in thesecond dimension with 12.5% polyacrylamide gels. For the two-dimensionalelectrophoreses, the proteins in the extracts in TENP40 buffer wereprecipitated with ethanol, collected by centrifugation and dissolved in50 mM Tris-HCl, pH 7.4; 8 M urea and 0.5% β-mercaptoethanol. The markerproteins as well as the two-dimensional electrophoresis standards (2-DSDS-PAGE standards™) from Bio-Rad were used as molecular weight markersor as isoelectric point references.

After electrophoresis, the proteins were stained with Coomassie blue orwith the aid of silver nitrate (silver stain plus kit Bio-Rad Lab.), ortransferred to a reinforced nitrocellulose membrane (Schleicher &Schuell, Dassel, Germany). The membranes were then stained with Ponceaured or with Protogold (British BioCell International, Cardiff, UK) andimmunodetected with monoclonal antibodies, as described above. Thereactivities were revealed with the ECL™ kit from Amersham (ECL™ Westernblotting kit, Amersham International, Aylesbury, UK), according to themanufacturer's protocol.

Results

The polypeptide of 52-56 kDa was detected in the extracts in TE and TEUbuffers (starting from a urea concentration equal to 6 M), but not inthe sequential extracts in TENP40 and TUDTT buffers. Furthermore, if theextract in TE buffer was centrifuged for 30 minutes at 100,000×g, thepolypeptide was completely present in the supernatant. The monoclonalantibody G36-19 also recognized several polypeptides of lower apparentmolecular weight, which were only partially extracted in the presence ofurea (even at the concentration of 8 M), and were partially extracted inthe presence of a reducing agent. Whether in TE buffer or in thepresence of urea, no polypeptide was immunodetectable in the last of thethree extracts produced. This indicates that the extraction was completein each of the steps. The control experiments carried out in the absenceof primary antibody or with the antibody MOPC-21 always proved to benegative. It is probable that the immunodetected forms of polypeptidesof low molecular weight were not products of degradation generatedduring the extraction steps, since their proportion did not vary fromone preparation to another and since the extractions were always carriedout in the presence of protease inhibitors.

Human Corneodesmosine is a Component of the Horny Envelopes

In order to confirm, at the biochemical level, that the corneodesmosineis covalently linked to the horny envelopes, fragments, generated byproteolysis of envelopes highly purified from human plantar epidermis,were analysed with the monoclonal antibody G36-19.

Preparation and Analysis of the Horny Envelopes

Human horny envelopes were purified from the plantar horny layer andmammary epidermis, as described above. Briefly, the samples wereextracted by repeated boiling, with vigorous stirring, in a solutioncontaining 2% SDS (w/v) and dithiothreitol at the concentration of 25mM, and than at 37° C. for 72 hours in a solution containing urea at theconcentration of 8 M and dithiothreitol at the concentration of 25 mM.The envelopes extracted in urea were sedimented and then suspended in asolution containing 0.1% SDS, glycine at the concentration of 192 mM andTris at the concentration of 125 mM. Finally, they were electrodialysedagainst this same buffer for 72 hours. The purified horny envelopes werethen collected by centrifugation, washed with distilled water andcounted. They were analysed morphologically, and then by indirectimmunofluorescence, as described in the prior art. Digestion withprotease V8 and immunodetection of the proteolysis products were carriedout as described in the prior art.

Results

The envelopes were incubated for increasing periods with protease V8,and the fragments produced were separated by SDS-PAGE andimmunodetected. The monoclonal antibody G36-19 strongly labelled a largenumber of bands having an apparent molecular weight greater than 50 kDa,some of which were located at the upper limit of the gel. This indicatesthat the corneodesmosine is incorporated inside the large-sizedheteropolymeric structures resulting from the proteolysis of theenvelope. Several bands stained with Protogold were not immunodetected,which confirms the specificity of the reaction. No band was clearlyimmunodetected with the monoclonal antibody G36-19 in the absence ofproteolysis, which proves the existence of covalent bonds betweencorneodesmosine and other constituents of the horny envelopes.

None of the fragments produced by digestion of horny envelopes purifiedfrom mammary epidermis was immunodetected by the monoclonal antibodyG36-19. In agreement with this result, these envelopes were a lot lesslabelled, and only at their periphery, when they were analysed with themonoclonal antibody G36-19 in indirect immunofluorescence.

Human Corneodesmosine of 52-56 kDa is a Basic Phosphoprotein

To determine the isoelectric point of corneodesmosine, human epidermiswas extracted directly in a buffer containing a detergent (TENP40buffer), and the proteins extracted were separated by two-dimensionalelectrophoresis (NEpHGE/SDS-PAGE) and immunodetected with the monoclonalantibody G36-19. The corneodesmosine of 52-56 kDa showed a basicisoelectric point greater than 8. This result was confirmed byimmunodetection after IEF/SDS-PAGE separation. The proteinimmunodetected by the monoclonal antibody G36-19 was not visible afterCoomassie blue staining, even when 100 μg of proteins in the extract inTENP40 buffer were loaded on the gel. This therefore suggests that thecorneodesmbsine of 52-56 kDa is a quantitatively minor protein,representing less than 0.1% of the proteins extracted. Furthermore, theimmunoreactive protein migrated in the form of several juxtaposed spots,suggesting the presence of post-translational modifications. To analysethe possible phosphorylation of human corneodesmosine, the latter wasaffinity-purified on a Sepharose matrix (HiTrap-NHS, Pharmacia)activated with N-hydroxysuccinimide groups and then coupled with themonoclonal antibody G36-19. Next, it was analysed by immunotransfer withantibodies specific for phosphoserine or for phosphotyrosine. One of thefive anti-phosphoserine monoclonal antibodies used immunodetectedcorneodesmosine in a specific manner. In contrast, neither themonoclonal antibody PY20 nor an antiserum, directed againstphosphotyrosine, recognized corneodesmosine.

Human Corneodesmosine of 52-56 kDa is a Glycoprotein

Affinodetection with Lectins

The corneodesmosine of 52-56 kDa was partially purified from an extractin TENP40 buffer by affinity chromatography. The bound corneodesmosinewas eluted with 3% SDS, deposited on an electrophoresis gel andtransferred onto nitrocellulose membranes. The membranes were incubatedin a blocking buffer, and then with biotinylated lectins sold by PierceChemical Co., diluted to 1 μg/ml: agglutinin from Pisum sativum (PSA),from wheat germ (WGA), from Ricinus communis (RCA), from Dolichosbifluorus (DBA) and from Arachis hypogaea (PNA). After rinsing, thelectins were detected with streptavidin labelled with peroxidase(diluted 1/400,000), and the ECL™ kit from Amersham, as described above.

Deglycosylation Experiments

The extract in TENP40 buffer (10 μg) was kept boiling for 3 minutes in20 μl of a sodium phosphate buffer at pH 7.2 containing 1% (w/v) of SDS.Nonidet P40 and EDTA were added in order to obtain a final concentrationof, respectively, 1% and 20 mM. 2.4 units of N-glycosidase F (EC3.2.2.18, Boehringer Mannheim) were added and the reaction mixture wasincubated at 37° C. for 6 hours. The proteins (34 μg) of an extract inTENP40 buffer were also incubated with 5 mU ofendo-α-N-acetylgalactosaminidase (EC 3.2.1.97) at 37° C. for 6 hours, inthe presence or otherwise of N-glycosidase F or neuraminidase (EC3.2.1.18), under the conditions described by the manufacturer (OxfordGlycoSystems Ltd., Abingdon, UK). The reactions were stopped by 2minutes of boiling in sample buffer. The deglycosylation of fetuin, usedas positive control, was tested by SDS-PAGE and with the biotinylatedlectins.

The treated and untreated samples were separated by electrophoresis andanalysed by immunodetection and affinodetection, as described above.

Chromatography on Concanavalin A-Sepharose

An extract in TENP40 buffer was directly injected, with a flow rate of0.5 ml/minute, into a Concanavalin A Sepharose 4B column (ConASepharose, Sigma) which had been previously equilibrated with washingbuffer: 20 mM Tris-HCl, pH 7.4, containing 0.2 M NaCl. After washing,with a flow rate of 1 ml/minute, with 15 ml of this buffer, the adsorbedproteins were eluted with a flow rate of 0.5 ml/minute withmethyl-α-D-mannopyranoside (Sigma) diluted to 0.5 M in washing buffer.The proteins were then separated by SDS-PAGE and analysed as describedabove.

Results:

The proteins of an extract in TENP40 buffer, containing the 52-56 kDacorneodesmosine, were treated with various glycosidases and analysed inimmunotransfer with two monoclonal antibodies directed against thepolypeptide. The staining of the total proteins of the extract did notshow apparent degradation during the incubation. The treatment withN-glycosidase F induced a reduction of about 5 kDa in the apparentmolecular weight of the corneodesmosine. This result very stronglysuggests an N-glycosylation. In contrast, the treatments withendo-α-N-acetyl-galactosaminidase and/or neuraminidase did not modifythe migration of corneodesmosine. To confirm this result, humancorneodesmosine was affinity-purified and analysed with the aid ofbiotinylated lectins. Wheat germ agglutinin (WGA) was strongly bound tothe purified protein, unlike the other lectins tested which did not orvery weakly recognized it. Furthermore, corneodesmosine bound toConcanavalin A coupled to a Sepharose matrix. It was possible to eluteit with methyl α-D-mannopyranoside, a sugar which is specific for thislectin, at the concentration of 0.5 M, which proves the specificity ofthe binding.

Purification of Corneodesmosine:

After dermoepidermal cleavage, the epidermis was homogenized in TEAbuffer: 40 mM Tris-HCl, pH 7.5; 10 mM EDTA; 10 μg/ml aprotinin and4-(2-aminoethyl) benzenesulphonyl fluoride (Interchim, Paris, France)used at 0.8 mM. The homogenate was centrifuged for 15 minutes at 15,000g. The supernatant was clarified by filtration on filters whose porediameter is 0.45 μm (Puradisc™25 AS; Whatman, Clifton, N.J.), andinjected with a flow rate of 0.3 ml/minute on an anion-exchange column(Hi Trap Q, Pharmacia LKB) which had been equilibrated in the washingbuffer: 20 mM Tris-HCl, pH 7.5. The proteins not retained (that is tosay about 5% of the total proteins of the extract) were directlyinjected at the same flow rate into an affinity column prepared asfollows: about 2 mg of monoclonal antibody F28-27 were coupled to 1 mlof Sepharose 4B matrix activated by N-hydroxysuccinimide groups(HiTrap-NHS), as is recommended by the manufacturer Pharmacia LKB. Thecolumn was washed exhaustively with a flow rate of 1 ml/minute with thewashing buffer in the presence and then in the absence of 1 M NaCl. Theimmunoadsorbed proteins were eluted with a flow rate of 0.3 ml/minutewith 0.2 M glycine at pH 2.5; the pH of the eluted fractions wasimmediately neutralized with Tris base at a concentration of 2 M. Theproteins of the different fractions were analysed in immunotransfer withthe monoclonal antibody G36-19, or with the control monoclonal antibodyMOPC-21. The fractions containing the proteins thus eluted were mixed,and then lyophilized. The proteins of the lyophilizate were analysed asdescribed above, by one-or two-dimensional electrophoresis. Thecorneodesmosine was then cut out of the gel so as to be sequenced.

Results

The corneodesmosine of 52-56 kDa was purified by an anion-exchangechromatography followed by affinity chromatography. The entirecorneodesmosine extracted is specifically bound to the affinity column,from which it was possible for it to be eluted in the presence ofglycine. The staining with Protogold of the transferred proteins showsthe high degree of purification obtained, since the corneodesmosine ishighly predominant in these fractions. Similar results were obtainedwhen the affinity chromatography was carried out on a matrix coupledwith the monoclonal antibody F28-27 or with the monoclonal antibodyG36-19. A two-dimensional gel analysis confirmed the basic isoelectricpoint of the purified corneodesmosine and showed that it is the onlyprotein eluted having an apparent molecular weight of 52-56 kDa. Thecorneodesmosine thus purified was separated by electrophoresis and thegel band containing this protein was cut out and then the protein wassequenced.

Instability of the Purified Polypeptide

During its numerous trials to purify corneodesmosine, the applicantencountered two major problems: (1) the low proportion of this proteinamong the epidermal proteins and (2) its instability, possibly due toits high sensitivity to the action of proteases.

Different tests were used to try to stabilize the corneodesmosine:addition of zinc (inhibitor of the chymotryptic enzyme of the hornylayer), reducing agents (β-mercaptoethanol or dithiothreitol),denaturing agents (SDS), glycerol, inhibitors specific for deriseproteases (PMSF, aprotinin) or a cocktail of inhibitors of variousproteases (aprotinin, leupeptin, pepstatin, benzamidine, phenanthroline,PMSF) or suppression of the freezing/thawing steps. Finally, the absenceof freezing/thawing steps, the presence of 2% SDS or the presence of thecocktail of inhibitors, and preferably a combination of these conditionsproved to be the most effective for stabilizing corneodesmosine.

Sequencing of the Purified Polypeptide

For the sequencing, the bands which correspond to corneodesmosine or tofragments of corneodesmosine (52-56 kDa, 45 kDa), identified on theelectrophoresis gels were cut out. The proteins were then directlydigested in the gel with the endopeptidase lys-C (EC 3.4.99.30) and thepeptides generated were purified by HPLC using a DEAE-C18 column. Thepeptides thus selected were sequenced by the method of Edman degradationcycles on a Procise Sequencer apparatus from the company AppliedBiosystems, according to the supplier's instructions.

Results

No sequencing from the NH₂-terminal end was possible, suggesting theprotection of the protein at this end.

Internal sequencings made it possible to establish the sequence of twofragments, namely:

A: Lys Ser Tyr Gly Gly Tyr Glu Val Val Gly Gly Ser Ser Asp Ser Gly

B: Lys Ile Tyr Pro Val Gly Tyr Phe Thr Lys

This result confirms that obtained by the molecular biology techniqueswhich allowed the deduction of the amino acid sequence of thepolypeptide of the invention from the nucleotide sequence.

Construction of a Human Epidermis Expression Library

The cloning of the cDNA for human corneodesmosine could not be carriedout using preexisting expression libraries. Indeed, the latter wereproduced either from mouse epidermis, or from human keratinocytescultured in a monolayer, conditions which do not allow the expression ofthe genes characteristic of epidermal terminal differentiation. Theapplicant therefore constructed a human epidermal expression library.

Extraction of Poly(A)⁺ Ribonucleic Acids (RNA) From Human Epidermis

It was carried out using samples of mammary skin, surgical wasteobtained after plastic surgery. The subcutaneous adipose tissue wasdissected and then the skin pieces were cut up with the aid of adermatome and incubated for 2 hours at 37° C., in a trypsin solution(solution A, Gibco BRL), epidermal face facing upwards. Thedermo-epidermal cleavage was then carried out with tweezers. Theepidermal sheets obtained were rinsed in phosphate buffer (PBS, pH 7.4).The extraction of the poly(A)⁺ RNAs was carried out according to theprotocol proposed in the kit “mRNA purification kit” marketed by Dynal(Oslo), after homogenization of the sheets with the aid of a “Turax”,directly in the buffer provided (lysis/binding buffer). The principle ofthis kit uses the affinity of the messenger RNA (mRNA) containing apoly(A) end for the magnetic beads coated with oligo(dT)₂₅. After 5minutes of incubation at room temperature in the lysate, the beads wereisolated with a magnet and subjected to 3 washes. The poly(A)⁺ RNAseluted by incubating the beads at 65° C. for 2 minutes in a 2 mM EDTAsolution, pH 8, were assayed using the “DNA DipStick” colorimetric kitmarketed by Invitrogen (San Diego, Calif.).

Construction of the Library

The library was constructed with the kit “ZAP Express cDNA Gigapack IIGold cloning kit” marketed by Stratagene (La Jolla, Calif.), followingthe protocol proposed by the supplier. The complementary DNAs (cDNA)were synthesized from 2 μg of poly(A)⁺ RNA, with the Moloney murineleukaemia virus reverse transcriptase (MMLV-RT), using oligo(dT)18primers comprising a restriction site for the enzyme XhoI and in thepresence of 5-methylcytidine triphosphate. The synthesis of the secondstrand was carried out by E. coli DNA polymerase I, and the ends of thecDNAs were made blunt with recombinant pfu polymerase. After addition ofEcoRI adaptors (T4 DNA ligase), and phosphorylation of the 5′ ends (T4polynucleotide kinase), the cDNAs were subjected to digestion with theenzyme XhoI. The selection of the cDNAs having a size greater than 500base pairs was carried out with a Sephacryl S-500 column. The ligationof the cDNAs with the two arms of the ZAP Express phage was carried outwith T4 phage ligase. The encapsulation of the recombinant phage wascarried out with the extracts “Gigapack II Gold Packaging Extract”provided by Stratagene. The strain XL-1 blue MRF′ of E. coli was usedfor the titration and the spreading of the library.

Results

This library was prepared from 2 μg of mRNA, extracted from a fragmentof human epidermis within the hours following its collection, byunidirectional cloning into the ZAP Express phage. It consists of about2×10⁵ independent clones.

Cloning of the Complementary Deoxyribonucleic Acid (CDNA) EncodingCorneodesmosine

Immunological Screening

The immunological screening, carried out without a prior amplificationstep, was performed on nitrocellulose membranes (Schleicher & Schuell,Dassel, Germany), incubated in anisopropyl-1-thio-β-D-galacto-pyranoside (IPTG, Stratagene) solution at10 mM for 10 minutes, dried and incubated for 4 h at 37° C. on thespread library. The immunological screening procedure was carried out asdescribed in the prior art with a cocktail of the three monoclonalantibodies G36-19, F28-27 and B17-21, used at the respectiveconcentrations of 0.2, 0.2 and 2 μg/ml. The positive clones wereisolated by conventional techniques and tested with each of the threemonoclonal antibodies separately.

Sequencing

The ZAP Express phages corresponding to the purified clones weresubjected to excision in vivo, with the aid of an ExAssist phage, asindicated by Stratagene. The plasmids obtained were amplified with theQiagen kit (Hilden, Germany), and then sequenced at the ends of theinserts, with primers T3 and M13 (Stratagene), using a Perkin-Elmer kit(ABI PRISM Dye Terminator cycle sequencing kit, Perkin Elmer, Fostercity, Calif.). Comparison of the sequences obtained with internationaldatabases was carried out with the Blast program.

Results

Given that the library was not amplified beforehand, the screening madeit possible to isolate 5 independent clones. The human cDNA fragmentscontained in the clones were partially sequenced from the ends, whichmade it possible to show that they were all overlapping and thereforeall encoded fragments of the same protein. The 5 clones were also testedwith each of the three monoclonal antibodies separately. Three clones(1.1, 4.4 and 5.1) proved to be reactive with the three monoclonalantibodies, the clone 5.5 with two monoclonal antibodies (F28-27 andG36-19) and finally the clone 1.2 was recognized only by the monoclonalantibody F28-27. These results conclusively demonstrate that the threemonoclonal antibodies recognize the same protein, thus confirming whathad been strongly suggested by previous biochemical studies. Theseresults also make it possible to order on the molecule the epitopesrecognized by each of the monoclonal antibodies. Indeed, given that theclones were constructed by reverse transcription starting from the 3′end of the mRNAs, which corresponds to the COOH end of the protein, itis possible to conclude that the epitope recognized by F28-27 is closestto this end, followed by the epitope for G36-19 and finally that forB17-21. Enzymatic restriction analysis of the size of these differentcDNA fragments confirmed that the 3 clones recognized by the 3monoclonal antibodies have the longest sequences (from 2 to 1.5 kb),whereas that which only F28-27 recognizes is the shortest (1 kb), thelast, recognized by 2 monoclonal antibodies, having an intermediate sizeof 1.3 kb.

Accordingly, all or part of the cDNA encoding human corneodesmosine wascloned, a protein characterized by the 3 monoclonal antibodies B17-21,G36-19 and F28-27 which recognize different epitopes, ordered on theprotein in the order indicated, from the NH₂ end to the COOH end.

The partial sequences of each clone, compared with the sequencesincluded in the international databanks, revealed a 99% identity withthe sequence of the S gene, registered in the GenBank databank under thenumber L20815. This analysis also showed that among the 5 clonesobtained, 4 correspond to the short form of the mRNA, only one, clone5.1, corresponding to the long form. These forms are derived from analternative choice of the polyadenylation site at the level of thenoncoding 3′ part of the primary transcript. This analysis made itpossible to specify the location of the epitopes recognized by our 3monoclonal antibodies, the epitope recognized by B17-21 is located inthe part of the protein corresponding to nucleotides 594 to 762, G36-19and F28-27 being respectively located in the 762-1044 and 1044-stopcodon zone. Finally, the clone 1.1, which contains the longest codingsequence, starts at nucleotide 348.

Cloning of the Complete cDNA Encoding Corneodesmosine

The preceding analyses showed that the clones obtained did not cover thecDNA part extending from nucleotide 1 to nucleotide 347. The applicanttherefore had to clone this missing part.

80 ng of poly(A)⁺ RNA extracted from human epidermis as described abovewere subjected to reverse transcription starting with random primers,using the kit “SuperScript” marketed by Gibco BRL. The amplification ofthe 5′ end of the cDNA for corneodesmosine was carried out starting with{fraction (1/20)} of the first strand synthesis reaction, with,upstream, an oligonucleotide corresponding to the sequence 2-20(GenBankL20815) and also comprising an SpeI adapter, and, downstream, anoligonucleotide complementary to the sequence 1017-1035. The polymerasechain reaction (PCR) conditions were the following: 3 minutes at 94° C.followed by 35 cycles with 80 sec at 94° C., 80 sec at 57° C. and 2minutes at 72° C. The single fragment obtained, purified afteragarose-TBE gel electrophoresis, digested with SpeI and EcoNI(nucleotides 2 to 1003), was cloned into the plasmid pBK-CMV-1.1(isolated by screening of the library) digested with the same enzymes,which made it possible to arrive at the plasmid ps11. The complete cDNAcontained in the plasmid ps11 was sequenced at least 3 times up tonucleotide 1700. Comparison with the sequence previously publishedrevealed four localized differences which were analysed at the genomiclevel.

Genomic Analysis of Corneodesmosine

Four localized differences were observed between the sequence of thecomplete cDNA isolated by the applicant and that of the S gene publishedin the prior art (Zhou Y. and Chaplin D. D., P. N. A. S. usa, Vol. 90,pp. 9470-9474, October 1993). These four localized differences wereanalysed by PCR, at the genomic level starting with ten control samplesof DNA extracted from human blood according to conventional methods.

Analysis of the possible insertion of a guanine at position 1514 wascarried out after PCR amplification of the genomic region betweennucleotides 1446 and 1786 (GenBank, L20815), carried out underconventional conditions. The DNA fragment obtained for each sample wassubjected to digestion with BsiMI on the one hand and NciI on the other.Electrophoresis on a 3% NuSieve GTG gel (FMC, Rockland, Me.) was carriedout in TBE buffer.

The study of the sequence at position 66 was carried out according tothe ASA (allele specific amplification) method. Two PCR reactions werecarried out in parallel with, downstream, an oligonucleotidecomplementary to the sequence 572-91 and, upstream, an. oligonucleotidecorresponding to the sequence 52-65 and ending at position 66 eitherwith. A or with T. The PCR reactions were carried out with 0.5 μg ofgenomic DNA, 2.5 pmol of each oligonucleotide, and 0.5 U of Taqpolymerase (Appligene). The amplification conditions are 94° C., 2minutes and then 30 cycles with 50 seconds at 94° C., 1 minute at 56° C.and 2.5 minutes at 72° C.

Analysis of the bases at position 1118 and 1236 was carried out afteramplification with a single oligonucleotide pair: sense oligonucleotide908-27, antisense 1573-93. The PCR conditions are the same as above,with a polymerization time limited to 1 minute. The fragments weresubjected to digestion with BsmI or Hin6 I for the respective study ofpositions 1118 and 1236 and then separated by electrophoresis on a 3%NuSieve gel.

Results

The insertion of a guanine at position 1514 causes a reading frame shiftand moves the stop codon from position 1523 to position 1603. Theprotein produced by this sequence differs at the level of the last twoamino acids (I and S) which are replaced by the sequence:

Asp Ile Leu Ala Gln Val Lys Pro Leu Gly Pro Gln Leu Ala Asp Pro Glu ValPhe

Leu Pro Gln Gly Glu Leu Leu Asp Ser Pro

which corresponds to an addition of 27 amino acids relative to thepublished sequence. The 5. cDNAs previously, isolated all exhibited thisinsertion; the study was extended to other individuals. Ten controlsamples of human genomic DNA, obtained from ten individuals, wereamplified in this region and analysed by enzymatic restriction. Indeed,the published sequence corresponds to the presence of a BsiMIrestriction site at position 1510 which disappears in the sequencecomprising an additional guanine, whereas an NciI site appears at 1512.The results are the same for the 10 individuals, namely that only theNciI site is present. This means that in these ten individuals,corneodesmosine is produced in a longer form than the published form ofthe S gene.

Three other localized differences, of the substitution type, were foundbetween the clones and the published sequence. The analysis of the tencontrol genomic samples by PCR and enzymatic restriction or by ASA,showed that polymorphism of the gene is involved. The applicant hasidentified at position 66 a conservative mutation T/A (Leu-Met), atposition 1118 a polymorphism A/G, corresponding to a silent mutationAla-Ala and, finally, at 1236 a polymorphism T/G corresponding to anonconservative mutation Ser-Ala.

Production of Recombinant Corneodesmosine

The complete cDNA encoding corneodesmosine was isolated, in the form ofan Ecl136 I/NotI fragment of 2106 base pairs, from the plasmid pS11. Thesubcloning was carried out into the vector PCDNA amp (In Vitrogen)previously digested with EcoRV and NotI, leading to the plasmid pl4-9.

The in vitro translation of corneodesmosine was carried out with the kit“TNT T7 Quick coupled transcription/translation system” marketed byPromega starting with the plasmid p14-9. This kit combines in one stepthe transcription with T7 phage RNA polymerase with translation with arabbit reticulocyte lysate. These reactions were carried out with 0.6 μgof plasmid in a volume of 25 μl, according to the manufacturer'sinstructions.

Corneodesmosine was expressed in COS-7 cells after transfection with theplasmid p14-9. The cells were transfected according to the conventionalDEAE-dextran protocol, supplemented by the addition of chloroquine and aDMSO shock, using 1 μg of plasmid/3×10⁵ cells. After 48 h of expression,the cells are washed twice with PBS, pH 7.4, and lysed in a 40 mMTris-HCl buffer, pH 7.5, containing 10 mM EDTA and 0.5% NP40, andcontaining a cocktail of protease inhibitors (Pharmingen, Inc.). AfterSDS-PAGE separation and electrotransfer, the samples are immunodetected,as described above.

Results:

The new complete sequence of the protein predicts a molecular weight of51.45 kDa, in the absence of any post-translational modification. Thecorneodesmosine synthesized in vitro, in the absence of microsomalmembranes, migrates with an apparent molecular weight of about 60 kDa.This shows that this protein has an aberrant migration in SDS-PAGE gel.Comigration with the predominant epidermal form extracted in a low-ionicstrength buffer demonstrates that the latter corresponds to a fairlyextensively truncated form of the protein, all the more so since theapplicant has shown that this epidermal form is glycosylated.

The transfection of COS-7 cells with the plasmid 14-9 reveals animmunoreactive protein which also migrates in the region of 60 kDa. Thesame results were obtained with the human neuroepithelioma line CHP 126,transfected by electroporation. All these results strongly suggest thatin the epidermis, corneodesmosine undergoes, prematurely and during itsmaturation, a specific proteolysis.

2 1 529 PRT Human 1 Met Gly Ser Ser Arg Ala Pro Trp Met Gly Arg Val GlyGly His Gly 1 5 10 15 Met Met Ala Leu Leu Leu Ala Gly Leu Leu Leu ProGly Thr Leu Ala 20 25 30 Lys Ser Ile Gly Thr Phe Ser Asp Pro Cys Lys AspPro Thr Arg Ile 35 40 45 Thr Ser Pro Asn Asp Pro Cys Leu Thr Gly Lys GlyAsp Ser Ser Gly 50 55 60 Phe Ser Ser Tyr Ser Gly Ser Ser Ser Ser Gly SerSer Ile Ser Ser 65 70 75 80 Ala Arg Ser Ser Gly Gly Gly Ser Ser Gly SerSer Ser Gly Ser Ser 85 90 95 Ile Ala Gln Gly Gly Ser Ala Gly Ser Phe LysPro Gly Thr Gly Tyr 100 105 110 Ser Gln Val Ser Tyr Ser Ser Gly Ser GlySer Ser Leu Gln Gly Ala 115 120 125 Ser Gly Ser Ser Gln Leu Gly Ser SerSer Ser His Ser Gly Ser Ser 130 135 140 Gly Ser His Ser Gly Ser Ser SerSer His Ser Ser Ser Ser Ser Ser 145 150 155 160 Phe Gln Phe Ser Ser SerSer Phe Gln Val Gly Asn Gly Ser Ala Leu 165 170 175 Pro Thr Asn Asp AsnSer Tyr Arg Gly Ile Leu Asn Pro Ser Gln Pro 180 185 190 Gly Gln Ser SerSer Ser Ser Gln Thr Ser Gly Val Ser Ser Ser Gly 195 200 205 Gln Ser ValSer Ser Asn Gln Arg Pro Cys Ser Ser Asp Ile Pro Asp 210 215 220 Ser ProCys Ser Gly Gly Pro Ile Val Ser His Ser Gly Pro Tyr Ile 225 230 235 240Pro Ser Ser His Ser Val Ser Gly Gly Gln Arg Pro Val Val Val Val 245 250255 Val Asp Gln His Gly Ser Gly Ala Pro Gly Val Val Gln Gly Pro Pro 260265 270 Cys Ser Asn Gly Gly Leu Pro Gly Lys Pro Cys Pro Pro Ile Thr Ser275 280 285 Val Asp Lys Ser Tyr Gly Gly Tyr Glu Val Val Gly Gly Ser SerAsp 290 295 300 Ser Tyr Leu Val Pro Gly Met Thr Tyr Ser Lys Gly Lys IleTyr Pro 305 310 315 320 Val Gly Tyr Phe Thr Lys Glu Asn Pro Val Lys GlySer Pro Gly Val 325 330 335 Pro Ser Phe Ala Ala Gly Pro Pro Ile Ser GluGly Lys Tyr Phe Ser 340 345 350 Ser Asn Pro Ile Ile Pro Ser Gln Ser AlaAla Ser Ser Ala Ile Ala 355 360 365 Phe Gln Pro Val Gly Thr Gly Gly ValGln Leu Cys Gly Gly Gly Ser 370 375 380 Thr Gly Ser Lys Gly Pro Cys SerPro Ser Ser Ser Arg Val Pro Ser 385 390 395 400 Ser Ser Ser Ile Ser SerSer Ala Gly Ser Pro Tyr His Pro Cys Gly 405 410 415 Ser Ala Ser Gln SerPro Cys Ser Pro Pro Gly Thr Gly Ser Phe Ser 420 425 430 Ser Ser Ser SerSer Gln Ser Ser Gly Lys Ile Ile Leu Gln Pro Cys 435 440 445 Gly Ser LysSer Ser Ser Ser Gly His Pro Cys Met Ser Val Ser Ser 450 455 460 Leu ThrLeu Thr Gly Gly Pro Asp Gly Ser Pro His Pro Asp Pro Ser 465 470 475 480Ala Gly Ala Lys Pro Cys Gly Ser Ser Ser Ala Gly Lys Ile Pro Cys 485 490495 Arg Ser Ile Arg Asp Ile Leu Ala Gln Val Lys Pro Leu Gly Pro Gln 500505 510 Leu Ala Asp Pro Glu Val Phe Leu Pro Gln Gly Glu Leu Leu Asp Ser515 520 525 Pro 2 1590 DNA Human 2 atgggctcgt ctcgggcacc ctggatggggcgtgtgggtg ggcacgggat gatggcactg 60 ctgctggctg gtctcctcct gccagggaccttggctaaga gcattggcac cttctcagac 120 ccctgtaagg accccacgcg tatcacctcccctaacgacc cctgcctcac tgggaagggt 180 gactccagcg gcttcagtag ctacagtggctccagcagtt ctggcagctc catttccagt 240 gccagaagct ctggtggtgg ctccagtggtagctccagcg gatccagcat tgcccagggt 300 ggttctgcag gatcttttaa gccaggaacggggtattccc aggtcagcta ctcctccgga 360 tctggctcta gtctacaagg tgcatccggttcctcccagc tggggagcag cagctctcac 420 tcgggaagca gcggctctca ctcgggaagcagcagctctc attcgagcag cagcagcagc 480 tttcagttca gcagcagcag cttccaagtagggaatggct ctgctctgcc aaccaatgac 540 aactcttagc gcggaatact aaacccttcccagcctggac aaagctcttc ctcttcccaa 600 acctctgggg tatccagcag tggccaaagcgtcagctcca accagcgtcc ctgtagttcg 660 gacatccccg actctccctg cagtggagggcccatcgtct cgcactctgg cccctacatc 720 cccagctccc actctgtgtc agggggtcagaggcctgtgg tggtggtggt ggaccagcac 780 ggttctggtg cccctggagt ggttcaaggtcccccctgta gcaatggtgg ccttccaggc 840 aagccctgtc ccccaatcac ctctgtagacaaatcctatg gtggctacga ggtggtgggt 900 ggctcctctg acagttatct ggttccaggcatgacctaca gtaagggtaa aatctatcct 960 gtgggctact tcaccaaaga gaaccctgtgaaaggctctc caggggtccc ttcctttgca 1020 gctgggcccc ccatctctga gggcaaatacttctccagca accccatcat ccccagccag 1080 tcggcagctt cctcggccat tgcgttccagccagtgggga ctggtggggt ccagctctgt 1140 ggaggcggct ccacgggctc caagggaccctgctctccct ccagttctcg agtccccagc 1200 agttctagca tttccagcag cgccggttcaccctaccatc cctgcggcag tgcttcccag 1260 agcccctgct ccccaccagg caccggctccttcagcagca gctccagttc ccaatcgagt 1320 ggcaaaatca tccttcagcc ttgtggcagcaagtccagct cttctggtca cccttgcatg 1380 tctgtctcct ccttgacact gactgggggccccgatggct ctccccatcc tgatccctcc 1440 gctggtgcca agccctgtgg ctccagcagtgctggaaaga tcccctgccg ctccatccgg 1500 gatatcctag cccaagtgaa gcctctggggccccagctag ctgaccctga agttttccta 1560 ccccaaggag agttactcga cagtccataa1590

What is claimed is:
 1. A composition comprising, in a physiologicallyacceptable medium, one or more purified natural or syntheticpolypeptide(s), wherein each of said polypeptide(s) comprises the aminoacid sequence of SEQ ID NO:
 1. 2. The composition according to claim 1,wherein said polypeptide(s) are purified from mammals.
 3. Thecomposition according to claim 1, wherein said polypeptide(s) arepurified from mammalian skin.
 4. The composition according to claim 1,wherein said polypeptide(s) are purified from human skin.
 5. Thecomposition according to claim 1, wherein said polypeptide(s) arepurified from human epidermis.
 6. The composition according to claim 1,wherein said polypeptide(s) are involved in intercorneocyte cohesion. 7.The composition according to claim 1, wherein each of saidpolypeptide(s) comprising amino acid sequence (SEQ ID NO: 1) has anapparent molecular weight of between 50 and 60 kilodaltons, asdetermined by SDS-PAGE.
 8. The composition according to claim 1, whereinsaid polypeptide(s) are basic.
 9. The composition according to claim 1,wherein said polypeptide(s) are glycosylated.
 10. The compositionaccording to claim 1, wherein said polypeptide(s) are phosphorylated.11. The composition according to claim 1, wherein each of saidpolypeptide(s) is phosphorylated, basic and glycosylated and has anapparent molecular weight of between 50 and 60 kilodaltons, asdetermined by SDS-PAGE.
 12. The composition according to claim 1, whichcomprises a mixture of polypeptides derived from the proteolysis of saidpolypeptide(s).
 13. The composition according to claim 1 intended fortreating the thinning of the epidermis.
 14. The composition according toclaim 1 for treating trophic skin disorders or trophic skin disordersfollowing cicatrization disorders.
 15. A method for treating thethinning of the epidermis, comprising administering to the skin of asubject in need of treatment an effective amount of a compositioncomprising, in a physiologically acceptable medium, one or more purifiednatural or synthetic polypeptide(s), wherein each of said polypeptide(s)comprise the amino acid sequence of SEQ ID NO:
 1. 16. A method fortreating trophic skin disorders or trophic skin disorders followingcicatrization disorders comprising administering an effective amount ofa composition comprising, in a physiologically acceptable medium, one ormore purified natural or synthetic polypeptide(s), wherein each of saidpolypeptide(s) comprise the amino acid sequence of SEQ ID NO:
 1. 17. Acosmetic or pharmaceutical composition comprising, in a physiologicallyacceptable medium, a purified natural or synthetic polypeptidecomprising the amino acid sequence of SEQ ID NO:
 1. 18. A compositioncomprising, in a physiologically acceptable medium, a nucleic acidmolecule comprising the coding nucleotide sequence of SEQ ID NO:
 2. 19.The composition according to claim 1, wherein said polypeptide(s)strengthen intercorneocyte cohesion.
 20. The composition according toclaim 1, wherein said polypeptide(s) are specific to the horny andgranular layers.
 21. The composition according to claim 1, wherein saidpolypeptide(s) are specific to the structures involved in theintercorneocyte junction.
 22. The composition according to claim 1intended for treating excessive fragility of the skin.
 23. Thecomposition according to claim 1 intended for strengtheningintercorneocyte cohesion.
 24. The composition according to claim 1intended for inducing the thickening of the horny layer.
 25. A methodfor strengthening intercorneocyte cohesion, comprising administering tothe skin of a subject in need of intercorneocyte cohesion strengtheningan effective amount of a composition comprising, in a physiologicallyacceptable medium, one or more purified natural or syntheticpolypeptide(s), wherein each of said polypeptide(s) comprise the aminoacid sequence of SEQ ID NO:
 1. 26. A method of promoting desquamation tothe skin of a subject in need of desquamation promotion, comprisingadministering an effective amount of the composition in aphysiologically acceptable medium, one or more purified natural orsynthetic polypeptide(s), wherein each of said polypeptide(s) comprisethe amino acid sequence of SEQ ID NO:1.